Objective To investigate the effect of radioactive iodine therapy under thyroid hormone withdrawal in differentiated thyroid cancer patients on health-related quality of life. Methods Patients who were diagnosed with differentiated thyroid cancer after thyroidectomy were involved in this study. All of them were managed with thyroid hormone withdrawal. Health-related quality of life was assessed using the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire Core 30 and its thyroid cancer module at three different time points. Changes in health-related quality of life were evaluated by Wilcoxon and Kruskal–Wallis tests. Univariable logistic regression analysis was used to determine social-demographic and clinical factors associated with worse health-related quality of life. Results A total of 99 differentiated thyroid cancer patients were involved in this study. Changes in health-related quality of life at different time points showed that 1 month post-radioactive iodine treatment, an improvement in nausea and vomiting, insomnia and appetite loss was observed. Impairments of global health, role, cognitive and social function and problems of discomfort in the head and neck, voice concerns, dry mouth, fatigue, pain, dyspnea, thyroid fatigue, fear, tingling or numbness, joint pain and shoulder function increased after radioactive iodine treatment. Univariable logistic regression analysis demonstrated potential factors associated with worse health-related quality of life. Thyroid stimulating hormone and parathyroid hormone levels were more sensible to changes in functional domain. Patients aged ≥55-year-old, with annual income under ¥50 000, low parathyroid hormone and pT4 tumour stage experienced higher changes in symptom scales after radioactive iodine treatment. Conclusion After radioactive iodine treatment, differentiated thyroid cancer patients experienced negative health-related quality of life, and most of these impairments might not recover in the short term. Thyroid stimulating hormone and parathyroid hormone levels, annual income and pT tumours stage were independent risk factors for decreased health-related quality of life.